The present invention relates to the field of diagnostic and prognostic methods and kits, treatments, and compositions useful in understanding and identifying glaucoma, related intraocular pressure-disorders, and steroid sensitivity.
A group of debilitating eye diseases, the xe2x80x9cGlaucomasxe2x80x9d represent the leading cause of preventable blindness in the United States and other developed nations. In general, glaucomas are characterized by the alteration of the trabecular meshwork (TM), which consists of specialized endothelial cells and their associated connective tissue. The TM endothelial cells line the path the aqueous humor of the eye filters through during the normal, physiological flux. The cells generate and regulate the TM by producing extracellular molecules, the composition of which is thought to directly control the aqueous fluid flow.
In Primary Open Angle Glaucoma (xe2x80x9cPOAGxe2x80x9d), the most common form of glaucoma, an alteration in the TM leads to an obstruction of the normal ability of aqueous humor to leave its chamber surrounding the iris. However, the specific cells in the chamber between the iris and the cornea, in a region called the iridocorneal angle, remain xe2x80x9copenxe2x80x9d in that they continue to allow the egress of aqueous fluid (see, Vaughan, D. et al., In: General Ophthalmology, Appleton and Lange, Norwalk, Conn., pp. 213-230 (1992); and Gray""s Anatomy, 37th Ed., Churchill Livingstone, London, pp. 1180-1190 (1989)). As a result of the alteration in the TM and the obstruction, an increased intraocular pressure (xe2x80x9cIOPxe2x80x9d) can be observed. IOP can result in progressive visual loss and blindness if not treated appropriately and in a timely fashion.
Glaucomas are estimated to affect between 0.4% and 3.3% of all adults over 40 years old (Leske, M. C. et al., Amer. J. Epidemiol. 113:1843-1846 (1986); Bengtsson, B., Br. J. Ophthamol. 73:483-487 (1989); Strong, N. P., Ophthal. Physiol. Opt. 12:3-7 (1992)). Moreover, the prevalence of the disease rises to over 6% of those 75 years or older (Strong, N. P., Ophthal. Physiol. Opt. 12:3-7 (1992)).
A link between steroid, corticosteroid, or glucocorticoid treatments and the increased IOP found in POAG disease has long been suspected. While only 5% of the normal population have high IOP increases in response to topical glucocorticoids, greater than 40-50% of similarly treated patients with POAG show a high IOP increase (16 mm Hg). In addition, an Open Angle Glaucoma may be induced by exposure to glucocorticoids. This observation has suggested that an increased or abnormal glucocorticoid response in trabecular cells of the TM may be involved in POAG (Zhan, G. L. et al., Exper. Eye Res. 54:211-218 (1992); Yun, A. J. et al., Invest. Ophthamol. Vis. Sci. 30:2012-2022 (1989); Clark, A. F., Exper. Eye Res. 55: 265 (1992); Klemetti, A., Acta Ophthamol. 68: 29-33 (1990); Knepper, P. A., U.S. Pat. No. 4,617,299).
The ability of glucocorticoids to induce a glaucoma-like condition has led to efforts to identify genes or gene products induced by the cells of the trabecular meshwork in response (Polansky, J. R. et al., In: Glaucoma Update IV, Springer-Verlag, Berlin, pp. 20-29 (1991); Polansky J. R. and Weinrob, R. N., In: Handbook of Experimental Pharmacology, Vol. 69, Springer-Verlag, Berlin, pp. 461-538 (1984)). Initial efforts using short-term exposure to dexamethasone revealed only changes in specific protein synthesis. Extended exposure to relatively high levels of dexamethasone was, however, found to induce the expression of related 66 kD and 55 kD proteins that could be visualized by gel electrophoresis (Polansky, J. R. et al., In: Glaucoma Update IV, Springer-Verlag, Berlin, pp. 20-29 (1991)). The induction kinetics of these proteins as well as their dose response characteristics were similar to the kinetics that were required for steroid-induced IOP elevation in human subjects (Polansky, J. R. et al., In: Glaucoma Update IV, Springer-Verlag, Berlin, pp. 20-29 (1991)). Problems of aggregation and apparent instability or loss of protein in the purification process were obstacles in obtaining a direct protein sequence.
Nguyen et al., U.S. patent application Ser. No: 08/649,432, filed May 17, 1996, now U.S. Pat. No. 5,789,169, the entire disclosure of which is hereby incorporated by reference as if set forth at length herein, disclosed a novel protein sequence (the TIGR, trabecular meshwork inducible glucocorticoid response protein) highly induced by glucocorticoids in the endothelial lining cells of the human trabecular meshwork. Nguyen et al. also disclosed the cDNA sequence for that protein, the protein itself, molecules that bind to it, and nucleic acid molecules that encode it, and provided improved methods and reagents for diagnosing glaucoma and related disorders, as well as for diagnosing other diseases or conditions, such as cardiovascular, immunological, or other diseases or conditions that affect the expression or activity of the protein.
Because increased IOP is a readily measurable characteristic of glaucoma, the diagnosis of the disease is largely screened for by measuring intraocular pressure (tonometry) (Strong, N. P., Ophthal. Physiol. Opt. 12:3-7 (1992), Greve, M. et al., Can. J. Ophthamol. 28:201-206 (1993)). Unfortunately, because glaucomatous and normal pressure ranges overlap, such methods are of limited value unless multiple readings are obtained (Hitchings, R. A., Br. J. Ophthamol. 77:326 (1993); Tuck, M. W. et al., Ophthal. Physiol. Opt. 13: 227-232 (1993); Vaughan, D. et al., In: General Ophthamology, Appleton and Lange, Norwalk, Conn., pp. 213-230 (1992); Vernon, S. A., Eye 7:134-137 (1993)). Patients may also have a differential sensitivity to optic nerve damage at a given IOP. For these reasons, additional methods, such as direct examination of the optic disk and determination of the extent of a patient""s visual field loss are often conducted to improve the accuracy of diagnosis (Greve, M. et al., Can. J. Ophthamol. 28: 201-206 (1993)). Moreover, these techniques are of limited prognostic value. In some aspects, the present invention fulfills the need for improved diagnostic and prognostic methods.
The elevation of intraocular pressure (IOP) due to topical corticosteroids (and other routes of administration) is an important clinical problem that limits the clinical use of these effective anti-inflammatory agents. If not observed in sufficient time, the IOP elevation (especially in certain individuals who show the high end of steroid-induced IOP elevations) can result in optic nerve damage and permanent visual field loss, termed xe2x80x9csteroid glaucoma.xe2x80x9d Even patients taking inhaled, nasal, rectal, and facial steroids may be at risk. The present invention, in part, provides improved diagnostic agents, prognostic agents, therapeutic agents and methods that address this clinical problem.
The invention relates to nucleic acids, genes, proteins and cells that can be used in the treatment, diagnosis, prognosis, and identification of glaucoma, IOP-related disorders, or steroid sensitivity. The invention encompasses numerous agents, compositions, and methods, some of which are described by the objects and aspects of the invention detailed below. Others can be devised from the entire contents of this disclosure, and from the detailed description that follows.
In one aspect, the invention relates to nucleic acids comprising non-coding regions or promoter regions associated with the TIGR (trabecular meshwork inducible glucocorticoid response) gene of mammals. These nucleic acids can be used in identifying polymorphisms in the genomes of mammals and humans that predict steroid sensitivity or a susceptibility to glaucomas or diseases related to alterations in IOP. A number of diagnostic or prognostic methods and kits can be designed from these nucleic acids.
In one embodiment, the nucleic acids can be used to identify or detect a single base polymorphism in a genome. In other embodiments, two or more single base polymorphisms or multiple base polymorphisms can be identified or detected. The detection of a known polymorphism can be the basis for diagnostic and prognostic methods and kits of the invention. Various methods of detecting nucleic acids can be used in these methods and with the kits, including, but not limited to, solution hybridization, hybridization to microarrays containing immobilized nucleic acids or other immobilized nucleic acids, amplification-based methods such as PCR and the like, and an appropriate biosensor apparatus comprising a nucleic acid or nucleic acid binding reagent.
In another aspect, the invention relates to specific sequences and variants or mutants from the promoter or 5xe2x80x2 regulatory region of the human TIGR gene and nucleic acids incorporating these sequences, variants or mutants. The nucleic acids can be incorporated into the methods and kits of the invention, or used in expression systems, vectors, and cells to produce a protein or polypeptide of interest, or used in methods to identify or detect regulatory proteins or proteins that specifically bind to promoter or regulatory regions of the TIGR gene. While many of the examples below detail work from human tissue, other animals may be used as a source of the sequences.
In one embodiment of this aspect of the invention, for example, nucleic acids having the disclosed TIGRmt11 sequence variant, represented by the change at nucleotide 5113 in SEQ ID NO: 1, 3, or 34 from T to C, or the change in nucleotide 5117 in SEQ ID NO: 2 from T to C. The presence of sequence variant mt11 is linked to the high IOP response to steroid treatments and a nucleic acid incorporating the singe base substitution can be used to identify and determine individuals at risk for developing glaucoma from undergoing a steroid treatment therapy, or a progression from an ocular hypertensive state, or those with a steroid sensitivity. And, because of the link between high IOP responses to steroids and the later development of glaucoma, nucleic acids having the TIGRmt11 sequence variant may also be used to identify the risk of developing glaucomas, such as POAG. The identification of changes in IOP can be done by any known means, however, the xe2x80x9cArmalyxe2x80x9d criteria is preferred (see Armaly, M. F., Arch. Ophthalmol. 70:492 (1963); Armaly, M. F., Arch Ophtalmol. 75:32-35 (1966); Kitazawa, Y. et al., Arch. Ophthalmol. 99:819-823 (1981); Lewis, J. M. et al., Amer. J. Ophthalmol. 106:607-612 (1988); Becker, B. et al. Amer. J.Ophthalmol. 57:543 (1967), all of which are specifically incorporated herein by reference in their entireties).
An object of the invention is to provide a method for diagnosing glaucoma in a patient which comprises the steps: (A) incubating under conditions permitting nucleic acid hybridization: a marker nucleic acid molecule, said marker nucleic acid molecule comprising a nucleotide sequence of a polynucleotide that specifically hybridizes to a polynucleotide that is linked to a TIGR promoter, and a complementary nucleic acid molecule obtained from a cell or a bodily fluid of said patient, wherein nucleic acid hybridization between said marker nucleic acid molecule, and said complementary nucleic acid molecule obtained from said patient permits the detection of a polymorphism whose presence is predictive of a mutation affecting TIGR response in said patient; (B) permitting hybridization between said marker nucleic acid molecule and said complementary nucleic acid molecule obtained from said patient; and (C) detecting the presence of said polymorphism, wherein the detection of the polymorphism is diagnostic of glaucoma.
Another object of the invention is to provide a method for prognosing glaucoma in a patient which comprises the steps: (A) incubating under conditions permitting nucleic acid hybridization: a marker nucleic acid molecule, said marker nucleic acid molecule comprising a nucleotide sequence of a polynucleotide that specifically hybridizes to a polynucleotide that is linked to a TIGR promoter, and a complementary nucleic acid molecule obtained from a cell or a bodily fluid of said patient, wherein nucleic acid hybridization between said marker nucleic acid molecule, and said complementary nucleic acid molecule obtained from said patient permits the detection of a polymorphism whose presence is predictive of a mutation affecting TIGR response in said patient; (B) permitting hybridization between said marker nucleic acid molecule and said complementary nucleic acid molecule obtained from said patient; and (C) detecting the presence of said polymorphism, wherein the detection of the polymorphism is prognostic of glaucoma.
Another object of the invention is to provide marker nucleic acid molecules capable of specifically detecting TIGRmt1, TIGRmt2, TIGRmt3, TIGRmt4, TIGRmt5, TlGRmt11 and TIGRsv1.
Another object of the invention is to provide a method for diagnosing steroid sensitivity in a patient which comprises the steps: (A) incubating under conditions permitting nucleic acid hybridization: a marker nucleic acid molecule, the marker nucleic acid molecule comprising a nucleotide sequence of a polynucleotide that is linked to a TIGR promoter, and a complementary nucleic acid molecule obtained from a cell or a bodily fluid of the patient, wherein nucleic acid hybridization between the marker nucleic acid molecule, and the complementary nucleic acid molecule obtained from the patient permits the detection of a polymorphism whose presence is predictive of a mutation affecting TIGR response in the patient; (B) permitting hybridization between said TIGR-encoding marker nucleic acid molecule and the complementary nucleic acid molecule obtained from the patient; and (C) detecting the presence of the polymorphism, wherein the detection of the polymorphism is diagnostic of steroid sensitivity.
Further objects of the invention provide a nucleic acid molecule that comprises the sequence of SEQ ID NO: 1 or 34, recombinant DNA molecules containing a polynucleotide that specifically hybridizes to SEQ ID NO: 1 or 34 and substantially purified molecules that specifically bind to a nucleic acid molecule that comprises the sequence of SEQ ID NO: 1 or 34.
Further objects of the invention provide a nucleic acid molecule that comprises the sequence of SEQ ID NO: 3, recombinant DNA molecules containing a polynucleotide that specifically hybridizes to SEQ ID NO: 3 and substantially purified molecules that specifically bind to a nucleic acid molecule that comprises the sequence of SEQ ID NO: 3.
Additional objects of the invention provide a nucleic acid molecule that comprises the sequence of SEQ ID NO: 4, recombinant DNA molecules containing a polynucleotide that specifically hybridizes to SEQ ID NO: 4 and substantially purified molecules that specifically bind to a nucleic acid molecule that comprises the sequence of SEQ ID NO: 4.
Additional objects of the invention provide a nucleic acid molecule that comprises the sequence of SEQ ID NO: 5, recombinant DNA molecules containing a polynucleotide that specifically hybridizes to SEQ ID NO: 5 and substantially purified molecules that specifically bind to a nucleic acid molecule that comprises the sequence of SEQ ID NO: 5.
An additional object of the present invention is to provide a method of treating glaucoma which comprises administering to a glaucomatous patient an effective amount of an agent that inhibits the synthesis of a TIGR protein.
Indeed, the molecules of the present invention may be used to diagnose diseases or conditions which are characterized by alterations in the expression of extracellular proteins.